Dysregulated immune and metabolic pathways are associated with poor survival in adult acute myeloid leukemia with CEBPA bZIP in-frame mutations

Acute myeloid leukemia (AML) with CEBPA bZIP in-frame mutations (CEBPAbZIP-inf) is classified within the favorable-risk group by the 2022 European LeukemiaNet (ELN-2022). However, heterogeneous clinical outcomes are still observed in these patients. In this study, we aimed to investigate the mutation profiles and transcriptomic patterns associated with poor outcomes in patients with CEBPAbZIP-inf. One hundred and thirteen CEBPAbZIP-inf patients were identified in a cohort of 887 AML patients homogeneously treated with intensive chemotherapy. Concurrent WT1 or DNMT3A mutations significantly predicted worse survival in AML patients with CEBPAbZIP-inf. RNA-sequencing analysis revealed an enrichment of interferon (IFN) signaling and metabolic pathways in those with a shorter event-free survival (EFS). CEBPAbZIP-inf patients with a shorter EFS had higher expression of IFN-stimulated genes (IRF2, IRF5, OAS2, and IFI35). Genes in mitochondrial complexes I (NDUFA12 and NDUFB6) and V (ATP5PB and ATP5IF1) were overexpressed and were associated with poorer survival, and the results were independently validated in the TARGET AML cohort. In conclusion, concurrent WT1 or DNMT3A mutations and a dysregulated immune and metabolic state were correlated with poor survival in patients with CEBPAbZIP-inf, and upfront allogeneic transplantation may be indicated for better long-term disease control.

Recently, it was discovered that the favorable outcomes of patients with CEBPA mutations are confined to those with in-frame mutations in bZIP (CEBPA bZIP-inf ), no matter whether they have CEBPA sm or CEBPA dm [4,14].These findings impact the current 5th WHO Classification (WHO-2022) [15], 2022 ELN recommendations (ELN-2022) [16] and International Consensus Classification (ICC) [17], resulting in the changing of the category, "AML with biallelic mutations of CEBPA" into "AML with in-frame bZIP mutations of CEBPA."However, the 5-year event-free survival (EFS) was less than 50% [4], and the cumulative incidence of relapse was approaching 40% in this category [18].It remains largely unknown how to explain the heterogeneous outcomes in this so-called "favorable category" at the mutational and transcriptomic level.The identification of dysregulated pathways that are independent of the mutational background, may provide novel options for therapeutic targeting.
In this study, we aimed to investigate the prognostic relevance of mutations and transcriptomics in AML patients with CEBPA bZIP-inf from a large cohort of 887 unselected de novo non-M3 AML patients.Our study showed that patients with poor survival were characterized by enrichment of interferon (IFN) pathways and oxidative phosphorylation.These data provide insights into nongenetic heterogeneity that may guide precise risk stratification and hold therapeutic potential in AML with CEBPA bZIP-inf .The findings were also well-validated in the TARGET AML cohort.

METHODS AND MATERIALS Subjects
We consecutively enrolled 887 newly diagnosed de novo non-M3 AML patients who received standard chemotherapy at the National Taiwan University Hospital (NTUH).Diagnosis and classification of AML were made according to the 2022 ICC [12] and WHO-2022 [15].Detailed patient characteristics and treatment regimens were summarized in the Supplementary Data [19].This study was approved by the Institutional Review Board of the NTUH, and written informed consent was obtained from all participants in accordance with the Declaration of Helsinki (approval number: 201709072RINC and 202109078RINB).

Cytogenetics
Chromosomal analyses were performed as described previously [20].Karyotypes were classified using Medical Research Council (MRC)-defined risk groups [21].

Mutation analysis
Gene mutations were examined via targeted next-generation sequencing (NGS), using the TruSight myeloid sequencing panel (Illumina, San Diego, CA, USA), which included 15 full exon genes and 39 oncogenic hotspot genes.HiSeq platform (Illumina, San Diego, CA, USA) was used for sequencing with a median reading depth of 12000×.Owing to suboptimal sequencing sensitivity, we verified CEBPA mutations via Sanger sequencing.Analysis of FLT3-ITD was performed via polymerase chain reaction (PCR), followed by fluorescence capillary electrophoresis and that of KMT2A-PTD, by PCR followed by Sanger sequencing [22].

RNA-sequencing (RNA-seq) analysis
Bone marrow (BM) mononuclear cells from 136 AML patients (36 with CEBPA bZIP-inf , 11 with CEBPA nonbZIP-inf , 89 with wild type of CEBPA and normal karyotype, CEBPA wt ) were performed with RNA-seq.The purified RNA was used to prepare the sequencing libraries using the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer's recommendations.The qualified libraries were then sequenced on Illumina NovaSeq 6000 with 150 bp paired-end mode.Trimming of adaptor sequences and removal of reads of low quality were performed using cutadapt (v2.3).Quantified reads were aligned to the human genome (GRCh38.p12)by STAR (v2.6.1 a) [23], and then gene-level read counts were generated based on the annotations of Gencode (v28) [24].The read counts cross all samples were normalized using the trimmed mean of the M values method as implemented in the calcNormFactors function of edgeR package [25], and gene expression in terms of log 2 (CPM + 1) (counts per million reads) was computed for further analysis.We used limma to assess the differentially expressed genes (DEGs) between conditions [26].The linear model was fit to the normalized expression data using a design matrix contrasting patients with short and long EFS.Log2 fold changes (logFC) were computed, along with adjusted P values (false discovery rate, FDR) corrected for multiple hypothesis testing using the Benjamini-Hochberg (BH) method.
We used Gene Set Enrichment Analysis (GSEA) software to investigate systematic enrichments of CEBPA mutation-governed expressional profiles with gene sets curated in the Molecular Signatures Database (MSigDB) [27].The statistical significance of the degree of enrichment was assessed by a 1000-time random permutation test [28].

External datasets
The gene expression data of GSE15210, derived from Affymetrix HG-U133 oligonucleotide microarrays (n = 61, 7 with CEBPA dm , 8 with CEBPA sm , and 46 with CEBPA wt ), and the TCGA AML RNA-seq data generated by the Illumina HiSeq 2000 platform (n = 173) were downloaded for validation purposes [10,29].To evaluate the prognostic significance of mitochondria complexes in an independent AML patient cohort, RNA-seq, gene mutation and clinical data of the TARGET AML cohort (n = 493) were downloaded from the Genomic Data Commons Data Portal (https:// portal.gdc.cancer.gov/)[30].Data from patients in the AMLSG cohort, the UK-NCRI trials, the TCGA cohort and NTUH Yunlin Branch cohort with available molecular annotations were further used to validate the prognostic relevance of concurrent mutations in CEBPA bZIP-inf [29,31,32].

Statistical analysis
The discrete variables were compared using the chi-square tests, but if the expected values of contingency tables were smaller than 5, Fisher's exact test was used.Mann-Whitney U tests were used to compare continuous variables and medians of distributions.OS was measured from the date of first diagnosis to the date of last follow-up or death from any cause.EFS was measured from the date of diagnosis until treatment failure, relapse from first complete remission (CR1) or death from any cause, whichever occurred first [33].Multivariate Cox proportional hazard regression analysis was used to investigate independent prognostic factors for OS and EFS.A P value < 0.05 was considered statistically significant.Statistical analyses were performed in the SPSS 23 (SPSS Inc., Chicago, IL, USA) and R software 4.3.1 (R Foundation for Statistical Computing, Vienna, Austria).
Next, we studied the prognostic impact of concurrent mutations with the aim of risk-stratifying the patients with CEBPA bZIP-inf more precisely.Concurrent WT1 or DNMT3A mutations predicted significantly worse OS (WT1 mutation, median, 28.2 months vs. NR, P = 0.020 and DNMT3A mutation, median, 6.1 months vs. NR, P = 0.013) and a trend towards worse EFS (WT1 mutation, median, 10.6 vs. 138.2months, P = 0.064 and DNMT3A mutation, 3.3 vs. 138.2months, P = 0.083) in patients with CEBPA bZIP-inf (Fig. 2B, C).Importantly, the outcome of CEBPA bZIP-inf with concurrent WT1 or DNMT3A mutations was similar to the ELN-2022 intermediate-risk group in terms of OS (P = 0.251) and EFS (P = 0.220).However, GATA2, TET2, ELN-2022 defined adverse gene mutations, and FLT3-ITD did not influence CR1, relapse rate, EFS and OS (Supplementary Fig. 2).Allogeneic hematopoietic stem cell transplantation (allo-HSCT) in CR1 was associated with significantly longer EFS and a trend towards longer OS compared with post-remission chemotherapy (PR-CT) only in CEBPA bZIP-inf patients with DNMT3A or WT1 mutations (EFS, median, NR vs. 8.9 months, P = 0.025; OS, median, NR vs. 14.0 months, P = 0.066, respectively) (Fig. 2D).For CEBPA bZIP-inf patients without DNMT3A or WT1 co-mutations, allo-HSCT in CR1 was associated with similar OS (P = 0.413) and EFS (P = 0.168) compared with PR-CT (Supplementary Fig. 3 and Supplementary Table 3).Given the limited number of cohorts with available CEBPA bZIP-inf status, we validated our findings with datasets comprising patients with CEBPA dm .This decision was supported by evidences indicating that the majority (94.1% in the current study and 90% in the study by Taube F et al.) [4] of CEBPA dm can be classified as CEBPA bZIP-inf .Similarly, concurrent DNMT3A mutation was associated with significantly poorer OS (median, 16.3 months vs. not reached, P = 0.029) compared with DNMT3A wild-type cases in the validation cohort (Supplementary Fig. 4 and Supplementary Table 4).
Characteristics and clinical outcomes of patients with CEBPA bZIP-inf with relapse There were 39 patients with CEBPA bZIP-inf who experienced a relapse.Twenty-nine (82.9%) of 35 patients who underwent reinduction chemotherapy achieved second complete remission (CR2).Among the 6 patients refractory to re-induction chemotherapy, two patients were found to have GATA2 mutations, two patients had WT1 mutations, two patients had cytogenetic evolution, and one patient had central nervous system involvement.Notably, the presence of WT1 mutation was associated with a lower probability of attaining CR2 (50% vs. 92.6%,P = 0.031), and a trend towards inferior OS from the time of relapse (median, 2.8 vs. 26.5 months, P = 0.144).The median OS from the time of relapse was 23.4 months.Twentyone patients received subsequent allo-HSCT, including 14 patients (66.7%) in CR2.Notably, patients with allo-HSCT after relapse had significantly better OS compared with those without allo-HSCT (median, 124 vs. 13.1 months, P = 0.001).Furthermore, allo-HSCT in CR2 was associated with significantly better OS compared with allo-HSCT in other disease statuses (median, not reached vs. 19.9months, P = 0.044) (Supplementary Fig. 5 and Supplementary Table 5).
For validation purposes, targets of MYC and E2F were shown to be significantly enriched in the TCGA cohort when we compared the patients with CEBPA bZIP-inf (n = to CEBPA wt with intermediate-risk cytogenetics (n = 14) (Supplementary Fig. 6A).Since there is no other public RNA-seq cohort for patients with CEBPA bZIP-inf , we performed validation in a public dataset of patients with CEBPA dm .In GSE15210, a total of 83 genes were differentially expressed between CEBPA dm and CEBPA wt patients [10].Fifty-one (83.6%) out of 61 downregulated genes and 22 (100%) of 22 upregulated genes were also significantly differentially expressed in our RNA-seq data when comparing the transcriptomes between patients with CEBPA dm and CEBPA wt .The major downregulated genes belonged to the homeobox gene family (HOXA9, HOXA10, HOXA5, HOXA3, HOXA6, etc).The results confirmed the concordance of our RNA-seq data with others (Supplementary Fig. 6B).Association of dysregulated immune and metabolic pathways with short EFS in patients with CEBPA bZIP-inf Among the 39 patients with CEBPA bZIP-inf who had relapsed, only 8 (20.5%) had WT1 or DNMT3A mutations.Since concurrent mutations could only partly explain the poor clinical outcomes in CEBPA bZIP-inf , we hypothesized that, in addition to mutations, transcriptomic variation underpins a large proportion of functional variation.To elucidate some patients with CEBPA bZIP-inf still had poor clinical outcomes at the transcriptomic level, we compared the RNA-seq data between CEBPA bZIP-inf patients with short EFS (n = 16, EFS < 2 years) and long EFS (n = 20).There were 138 DEGs with |logFC|>0.585(absolute fold change >1.5 or <0.67) and P value < 0.05, which could separate the patients well on unsupervised clustering and PCA (Fig. 4A-C and Supplementary Table 7).This finding highlighted the possibility that some genes with lower fold changes were also important determinants of clinical outcomes.The MsigDB Hallmark gene sets showed significant enrichment of immune and metabolic pathways in CEBPA bZIP-inf with short EFS.The enriched immune pathways include IFN alpha and gamma response (NES 1.89, FDR 0.003; NES 1.52, FDR 0.027, respectively).The Reactome gene sets and Gene Ontology on biological process revealed an enrichment of IFN alpha signaling and type I IFN response (Fig. 4D, E and Supplementary Fig. 7A).STING1, IRF2, IRF5, OAS2, IFI35, and FADD were significantly overexpressed in patients with short EFS (Fig. 4F).Higher expression of STING1, IRF5, OAS2, IFI35, and FADD were associated with a trend towards shorter EFS in CEBPA bZIP-inf (Supplementary Fig. 8).To validate the results in an independent cohort, RNA-seq data from 20 CEBPA-mutated patients in the TARGET AML cohort were analyzed.Similarly, gene sets related to response to IFN were significantly enriched in those with shorter EFS (Supplementary Fig. 7B).
We additionally studied the genetic and transcriptomic heterogeneity within the CEBPA nonbZIP-inf group (n = 11).Concurrent GATA2 mutation was associated with a trend towards longer EFS, while TET2 mutation was associated with a trend towards shorter OS, albeit the small number of patients.There were 822 DEGs between CEBPA nonbZIP-inf patients with short and long EFS, of which 24 coincided with the DEGs that differentiated the outcomes in patients with CEBPA bZIP-inf .The detailed enriched pathways on GSEA were shown in Supplementary Fig. 10.
To address the question whether enrichment of interferon signaling and metabolic pathways were associated with shorter EFS in other AML subtypes, we analyzed RNA-seq data from 60 AML patients with NPM1 mutation and compared the transcriptomes between patients with short EFS (EFS < 2 years, n = 34) and those with long EFS (n = 26).Those with short EFS did not have a significant enrichment of metabolic pathways, such as oxidative phosphorylation (NES −1.06, FDR 0.35) and fatty acid metabolism (NES 0.67, FDR 0.97).However, we observed a negative enrichment of interferon signaling (NES -3.34, FDR 0) in those with short EFS (Supplementary Fig. 11).Although enriched interferon signaling pathways have previously been associated with unfavorable prognostic implications in the overall AML cohort [37], our findings suggested that the enriched pathways are context-dependent and differ across various AML subtypes.

DISCUSSION
The current study leverages a large cohort of AML to study the genomic and transcriptomic pattern that contribute to poor survival in AML with CEBPA bZIP-inf , a new disease category in the ELN-2022 and 2022 ICC.We identified WT1 and DNMT3A mutations, dysregulated immune and metabolic signatures that highly correlated with poor survival.We have generated a publicly available database for patients with CEBPA bZIP-inf that contains patient demographics, clinical survival data, RNA-seq, and NGS data, which serves as a resource for the AML research community.
The reports on the prognostic impact of concurrent mutations in CEBPA bZIP-inf have been re limited to GATA2, WT1, and TET2 [4,18].Mutant GATA2 is associated with improved survival, while WT1 and TET2 mutations are associated with a lower probability of survival [4].In our study, concurrent WT1 or DNMT3A mutations were associated with significantly poorer survival.We are the first to report the prognostic relevance of DNMT3A mutations in CEBPA bZIP-inf , and the result has been validated in external databases.However, due to the small number of DNMT3A comutant patients in both our cohort and the external databases, further investigation in a larger independent cohort is still warranted to validate these findings.Recently, it was discovered that CEBPA and DNMT3A cooperate to induce leukemogenesis.Mutant CEBPA interacts with the long splice isoform DNMT3A and increase the aberrant methylation especially on PRC2 target genes [38].We additionally found that CEBPA bZIP-inf patients with WT1 or DNMT3A mutations had improved survival if receiving allo-HSCT in CR1.In addition, WT1-mutated patients had a lower probability of attaining CR2 and displayed a trend towards inferior OS following relapse.These results highlight the importance of utilizing a personalized treatment approach in determining the appropriate timing of allo-HSCT within the ELN-2022 favorable category.
Since WT1 or DNMT3A mutations are presented in only 20.5% patients with CEBPA bZIP-inf who had relapsed, it's apparent that non-genetic drivers underpin a large proportion of functional variation in leukemia.Indeed, the RNA-seq revealed enrichment of IFN and metabolic pathways in those with short EFS.Cumulative evidences have supported the role of immune microenvironment in AML, and the activation of IFN-related pathways has been shown to have negative prognostic implications in AML [37,39].Chronic activation of the IFN signaling pathway actually creates an immune-suppressed environment and mediates resistance to various cancer treatments [40].In our study, enriched IFN-related pathways also correlated with a poorer survival.Patients with short EFS and long EFS expressed comparable amounts of leukemia-associated antigens CD34, CD123, and CD117 (Supplementary Fig. 12).This observation suggests that bulk BM RNA-seq could capture elements of the tumor immune microenvironment in addition to features of the leukemic cell compartment.
Another notable discovery is the dysregulated metabolism in those with short EFS.The results corroborate the findings that aberrant metabolic states are hallmarks of various types of cancer [41].Oxidative phosphorylation, involving mitochondria complexes I-V, has been reported to play a role in leukemia stem cell maintenance in myeloid leukemia [42,43].Genes in the mitochondrial complexes I and V were most prominently dysregulated and also correlated with survival in the TARGET AML cohort.It's noteworthy that many genes in the mitochondrial complexes had fold change less than 2, suggesting biologically relevant functions occur even at very low fold changes in RNA levels [44].
The use of bulk BM aspirates is a potential limitation of our analysis.Single-cell RNA-seq or mass cytometry will further elucidate which cell component makes the major The number of patients with CEBPA mutations was not adequate in the TARGET AML cohort, which potentially decreased the reliability of the external validation.Besides, we did not perform mechanistic studies to elucidate the detailed molecular pathways in CEBPA bZIP-inf .Further studies are needed to investigate whether targeting of these pathways could sensitize the leukemic cells and ultimately reduce relapse in these patients.
In conclusion, our results highlight the importance of the combination of genetic and transcriptomic approaches to fully elucidate the heterogeneities within a WHO-2022/ICC-defined entity.We identified WT1 mutations, DNMT3A mutations, dysregulated interferon and oxidative phosphorylation as negative prognosticators in patients with CEBPA bZIP-inf .We provide a cohort with complete patient demographics, clinical survival, NGS, and RNA-seq data that will nourish further research in the AML community.
Fig. 4 Differential transcriptional signatures between CEBPA bZIP-inf with short and long EFS.A Heat map of normalized gene expression values of the top upregulated and downregulated DEGs in patients with short EFS.The DEGs were listed in Supplementary Table 7. B PCA showing separate clustering of transcriptomes between CEBPA bZIP-inf AML with short and long EFS based on the top DEGs.C Volcano plot showing DEGs with genes downregulated (logFC<0) and upregulated (logFC>0), respectively, in CEBPA bZIP-inf patients with short EFS compared with long EFS.Important genes in the metabolic and immune pathways are highlighted.D Enriched and depleted MSigDB Hallmark and KEGG gene sets between patients with short and long EFS.E GSEA enrichment plots showing overrepresentation of IFN response and metabolic gene set signatures within the CEBPA bZIP-inf transcriptome.F, G Scatter plots illustrating the log2-transformed, CPM-normalized expression values in samples associated with short vs. long EFS for genes involved in IFN response (F) and oxidative phosphorylation (G).*P value < 0.05.H Kaplan-Meier plots for EFS for patients with low expression (blue lines) and high expression (red lines) of NDUFA12, NDUFB6, ATP5PB, ATP5IF1 at diagnosis.The mean expression value for the respective gene in all CEBPA bZIP-inf samples was used to discretize between low and high expression.I High expression of ATP5F1C was associated with significantly shorter OS and EFS in patients with CEBPA bZIP-inf .

Fig. 1
Fig. 1 Survival outcomes stratified by CEBPA mutation statuses.CEBPA bZIP-inf was associated with significantly better OS and EFS than CEBPA nonbZIP-inf and CEBPA wt .

Fig. 2
Fig. 2 Concurrent mutations and their prognostic significance in CEBPA bZIP-inf .A Frequencies of concurrent mutations in patients with different CEBPA mutation statuses.Mutations with a frequency of at least 1% in one subgroup were shown.*P < 0.05 between CEBPA bZIP-inf and CEBPA wt , **P < 0.05 between CEBPA bZIP-inf and CEBPA nonbZIP-inf .B, C Concurrent WT1 or DNMT3A mutations were associated with significantly shorter OS and a trend towards shorter EFS in patients with CEBPA bZIP-inf .D Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) in first complete remission was associated with significantly longer EFS and a trend towards longer OS than post-remission chemotherapy (PR-CT) in CEBPA bZIP-inf patients with WT1 or DNMT3A mutations.

Fig. 3
Fig. 3 Transcriptional signatures in patients with CEBPA bZIP-inf compared with CEBPA wt .A PCA showing a separation of clustering of transcriptomes between AML with CEBPA bZIP-inf and CEBPA wt .B Volcano plot showing DEGs with genes downregulated (logFC<0) and upregulated (logFC>0), respectively, in patients with CEBPA bZIP-inf compared with CEBPA wt .Multiple genes in the HOX family were downregulated in CEBPA bZIP-inf .C Heat map of normalized gene expression values of the top upregulated and downregulated DEGs.The DEGs were listed in Supplementary Table 6.D, E Enriched and depleted MSigDB oncogenic and Hallmark gene sets between CEBPA bZIP-inf and CEBPA wt .

Table 1 .
Comparison of clinical and laboratory features between patients with AML with different CEBPA mutational statuses.